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. 2007 Apr;81(8):4264-71.
doi: 10.1128/JVI.02629-06. Epub 2007 Feb 7.

The adenovirus E4 ORF3 protein binds and reorganizes the TRIM family member transcriptional intermediary factor 1 alpha

Mark A Yondola  1 , Patrick Hearing
Affiliations

The adenovirus E4 ORF3 protein binds and reorganizes the TRIM family member transcriptional intermediary factor 1 alpha

Mark A Yondola et al. J Virol. 2007 Apr.

Abstract

One of the most interesting functions attributed to the adenovirus early region 4 open reading frame 3 (E4 ORF3) protein is its reorganization of promyelocytic leukemia (PML) protein nuclear bodies. These normally punctate structures are reorganized by E4 ORF3 into tracks that eventually surround viral replication centers. PML rearrangement is an evolutionarily conserved function of E4 ORF3, yet its cause and functional relevance remain mysteries. The E4 ORF3 protein coimmunoprecipitates with the PML protein, yet E4 ORF3 still forms tracks in cells that lack PML. The PML protein is a member of a larger protein family termed tripartite motif (TRIM) proteins. TRIM proteins contain a tripartite domain structure in proximity to their N termini that consists of a RING finger domain, followed by one or two B box domains and a C-terminal coiled-coil domain (collectively termed the RBCC domain). The order and spacing of these domains are evolutionarily conserved and thought to mediate protein-protein interactions and other functions. We implemented a proteomic approach to isolate cellular proteins that bind to E4 ORF3. We identified a novel interaction between E4 ORF3 and another TRIM family member, transcriptional intermediary factor 1 alpha (TIF1alpha). TIF1alpha functions by recruiting coactivators and/or corepressors to modulate transcription. The interaction between E4 ORF3 and TIF1alpha was validated by coimmunoprecipitation and binding of recombinant proteins. Indirect immunofluorescence assays demonstrated that TIF1alpha is reorganized into track structures that contain PML upon E4 ORF3 expression. The RBCC domain of TIF1alpha is sufficient for E4 ORF3-induced rearrangement, and TIF1alpha reorganization is conserved across adenovirus serotypes.

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Figures

FIG. 1.
FIG. 1.
Ad5 E4 ORF3 protein binds TIF1α. (A) Recombinant E4 ORF3 protein was immobilized on chitin beads and used to isolate cellular binding proteins from uninfected HeLa cell lysates. Shown is a representative silver-stained 10% SDS-polyacrylamide gel. Lane 1, molecular weight standards (size markers are indicated on the left). Lanes 2 to 4, HeLa cell proteins that bound to the intein-CBD tag alone (lane 2), wild-type E4 ORF3 (lane 3), or the N82A mutant E4 ORF3 protein (lane 4). A prominent band present specifically in lane 3 (arrow) was identified by mass spectrometry as TIF1α. (B) Schematic diagrams of PML and TIF1α protein domain organization. R, RING finger domain; B, beta box domain; CC, coiled-coil domain; H, HP1 binding region; N, nuclear hormone receptor interaction domain; P, PHD domain; B, Bromo domain. The 3′ exons used for the expression of different PML isoforms are indicated by an open rectangle.
FIG. 2.
FIG. 2.
E4 ORF3 interacts with TIF1α in vivo and in vitro. (A) The wild-type but not the N82A mutant E4 ORF3 protein coimmunoprecipitates with TIF1α. HeLa cells were transfected with a vector for the expression of T7-tagged TIF1α 24 h prior to infection with Ad vectors that expressed either the wild-type or the N82A HA-tagged mutant E4 ORF3 proteins. Immunoprecipitations were performed using anti-T7 antibody, and Western blots were probed using antibodies against T7 (left panel) and HA (right panel). Lanes 1 and 2, immunoprecipitated T7-TIF1α, indicated by an arrow. Lanes 3 and 4, wild-type and N82A mutant proteins present in the soluble extract prior to immunoprecipitation. Lanes 5 and 6, wild-type and N82A mutant proteins present in the T7 immunoprecipitate. The E4 ORF3 protein is indicated by an arrow. Antibody heavy and light chains are evident in lanes 5 and 6. (B) E4 ORF3 and TIF1α form a direct interaction. Recombinant wild-type and N82A mutant E4 ORF3 proteins were expressed in E. coli, coupled to chitin beads and incubated with an E. coli lysate containing recombinant GST-TIF1α. The beads were washed and bound GST-TIF1α was examined by Western blotting. Lane 1, input soluble GST-TIF1α. Lanes 2 and 3, GST-TIF1α present in the flowthrough of the wild-type and the N82A mutant E4 ORF3 incubations. Lanes 4 and 5, GST-TIF1α bound to the wild-type and the N82A mutant E4 ORF3 beads. Molecular size markers are shown, and GST-TIF1α is indicated by an arrow.
FIG. 3.
FIG. 3.
Wild-type E4 ORF3 protein is necessary for the reorganization of TIF1α into PML-containing tracks. The localization of endogenous TIF1α, with and without Ad infection, was examined by indirect immunofluorescence. (A) TIF1α localization (Alexa-546) in uninfected HeLa cells. (B to P) HeLa cells were infected with the following viruses: B to D, wild-type Ad5 dl309; E to G, mutant virus dl355; H to J, mutant virus inORF3; K to M, mutant virus dl355-N82A; N to P, mutant virus dl355. TIF1α localization (Alexa-546) was examined as shown in panels A, B, E, H, K, and N. E4 ORF3 localization (Alexa-488) was examined as shown in panels C, F, and L. Ad DBP localization (Alexa-350) was examined as shown in panel I. PML localization (Alexa-488) was examined as shown in panel O. Merge, panels D, G, J, M, and P.
FIG. 4.
FIG. 4.
E4 ORF3 is sufficient to reorganize TIF1α but does not reorganize TIF1β. (A) Endogenous TIF1β localization was examined in uninfected HeLa cells (Alexa-546). (B to D) HeLa cells were infected with mutant virus dl355, and TIF1β localization (Alexa-546) and E4 ORF3 localization (Alexa-488) were examined as shown in panels B and C, respectively. (D) Merge. (E to G) HeLa cells were transfected with a vector for the expression of wild-type HA-tagged E4 ORF3. After 24 h, endogenous TIF1α localization (Alexa-546) and E4 ORF3 localization (Alexa-488) were examined as shown in panels E and F, respectively. (G) Merge.
FIG. 5.
FIG. 5.
TIF1α reorganization by E4 ORF3 is conserved across Ad serotypes. HeLa cells were infected with the dl355-D105A/L106A mutant virus (A to C), the wild-type Ad4 virus (D to F), and Ad vectors expressing HA-tagged E4 ORF3 from Ad9 (G to I) or Ad12 (J to L). Endogenous TIF1α localization (Alexa-546) was examined as shown in panels A, D, G, and J. Ad4-infected cells were identified by staining for Ad DBP (Alexa-350) (E). E4 ORF3 localization (Alexa-488) was examined as shown in panels B, H, and K. (C, F, I, and L) Merge.
FIG. 6.
FIG. 6.
The N-terminal RBCC domain of TIF1α is sufficient for reorganization by E4 ORF3. A549 cells were transfected with vectors for the expression of EYFP-TIF1α wild-type and mutant proteins with EYFP fused to full-length TIF1α (A to D), EYFP fused to the TIF1α RBCC domain (E to H), and EYFP fused to TIF1α-ΔRBCC (I to L). EYFP-TIF1α localization was examined in uninfected cells (A, E, and I) and in cells infected with mutant virus dl355 (B, F, and J). E4 ORF3 localization was examined as shown in panels C, G, and K. (D, H, and L) Merge.
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